Field and outdoor hydroponic studies were conducted to determine the relative potential of soybeans (Glycine max L. Merr.) to utilize nitrate and atmospheric nitrogen as sources of nitrogen. Comparisons of nodulating and nonnodulating isollnes coupled with enzymatic assays of nitrate reductase (in vivo) and nitrogenase (in situ acetylene reduction) were used as indexes. Seasonal profiles indicate maximunmit rate utilization at the full‐bloom growth stage, with symbiotic N2(C2H2) fixation peaking some 3 weeks later during pod fill. Nitrogen fixation estimates based on comparison of nodulating and nonnodulating isolines are not valid under growth conditions of low nitrate levels because growth of the nonnodulated isoline is stunted and nitrate utilization is also impaired. Seed yield of plants totally dependent on atmospheric nitrogen was less than one‐half the yield of plants utilizing both nitrate and atmospheric nitrogen under hydroponic growth conditions. Plants grown on a low nitrate level had higher symbiotic N2(C2H2) fixation rates than those grown on no nitrate. Similarly, seed yield of plants grown in hydroponics on high nitrate levels, which inhibited symbiotic fixation, was less than yield of plants utilizing both nitrate and atmospheric nitrogen. Thus, both symbiotic N2 fixation and nitrate utilization appearede ssential for maximum yield.

Since N03-availability in the rooting medium seriously limits symbiotic N2 fixation by soybean (Glycine max [L.] Merr.), studies were initiated to select nodulation mutants which were more tolerant to N03-and were adapted to the Midwest area of the United States. Three independent mutants were selected in the M2 generation from ethyl methanesulfonate or N-nitroso-N-methylurea mutagenized Williams seed. All three mutants (designated NODI-3, NOD2-4, and NOD3-7) were more extensively nodulated (427 to 770 nodules plant-1) than the Williams parent (187 nodules plant1) under zero-N growth conditions. This Since the nts mutants selected by Gresshofls group were derived from the cultivar Bragg which is a Maturity Group VII line and cannot be grown to maturity under Midwest growing conditions, the current study was initiated to select nodulation mutants from a background which can be field tested in the major Midwest soybean production area. This paper provides the initial characterization of three soybean lines with enhanced nodulation capability which were selected from mutagenized populations of Williams (Maturity Group III). MATERIALS AND METHODS Plant MaterialSoybean (Glycine max [L.] Merr., cv Williams) seeds were mutagenized with EMS or NMU as previously described (17). In brief, the four mutagen-treatments (two chemicals and two postwash intervals) consisted of presoaking all seed for 16 h in vigorously aerated water, then treating separate seed lots for 2 h with 50 mM EMS or 5 h with 2.5 mm NMU, followed by either 5-or 9-h postwashes. The mutagenized seed (designated M,) was planted in the field and, at maturity, the four M2 seed lots were individually bulk harvested. The M2 seed lots were then screened for nodulation mutants either in the field or in greenhouse gravel beds.

A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase INAD(P)H-NR, pH 6.5; EC 1.6.6.21 activity from the nitric oxide and nitrogen dioxide (NO(X)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets.Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.51) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatographyanion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO(X) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent K. for nitrite of 0.49 millimolar, and an apparent K. for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO(X) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH2) and reduced methyl viologen (MV) can serve as electron donors for NO(X) evolution activity. The NADPH-, FMNH2-, and reduced MV-NO(,) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymercuribenzoate, whereas, the FMNH2 and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO(X). NADPH eluted both NR and NO(X) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate-or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean.It has been shown that the predominant compound evolved from soybean leaves during the purged in vivo NR3 assay is nitric oxide (NO) with trace amounts of nitrous oxide (N20) and 'Supported by an American Soybean Association research grant, project number 84953.

This study was conducted to determine by gas chromatography (GC) and mass spectrometry (MS) the identity and the quantity of volatile N products produced during the helium-purged in vivo NR assay of soybean (Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identification and quantitation was collected by cryogenic trapping of volatile compounds carried in the He-purge gas stream. As opposed to an earlier report, acetaldehyde oxime production was not detected by our GC method, and acetaldehyde oxime was shown to be much more soluble in water than the compound(s) evolved from soybean leaflets. Nitric oxide (NO) and nitrous oxide (N20) were identified by GC and GC/MS as the main N products formed. NO and N20 produced from soybean leaflets were both labeled with 5N when "5N-nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Other compounds co-trapped with NO and N20 were identified as air (N2, 02), C02, methanol, acetaldehyde, and ethanol. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N20 (13.9 and 0.37 micromole per gram fresh weight per 30 minutes, respectively) than did the soybean cv Williams (1.67 and 0.09 micromole per gram fresh weight per 30 minutes, respectively). In both species NO production was dominant. In contrast, with similar assays, NO and N20 were not evolved from leaves of the nrl soybean mutant which lacks the constitutive NR enzymes. In addition to soybean cv Williams, six other Glycine sp. examined evolved significant quantities of NO(x) (NO and NO2). Other species including Neonotonia wightii (Arn.) Lackey comb. nov., Pueraria montana (Lour.) Meff., and Pueraria thunbergiana Benth. evolved lower levels of NO(,).Klepper (5) demonstrated that herbicide treated soybean leaves form and release NO(X3 (thought to be predominately NO). He suggested that NO and NO2 were formed by a chemical reaction of NO2-(accumulated due to herbicide treatments) with plant metabolites, forming NO at low NO2-levels and NO and NO2 at higher NO2 levels. It was later shown (4) that N2 gas purging during the in vivo NR assay of soybean leaflets also resulted in the formation of NO(X) derived from NO2-accumulated during the assay. Harper's work (4) also indicated that the predominate ' Supported by an American Soybean Association research grant, project number 84953.2 Grateful recipient of a Wright Fellowship from the College of Agriculture, University of Illinois.3 Abbreviations: NO(X), refers collectively to nitric oxide (NO) and nitrogen dioxide (NO2); NR, nitrate reductase; DAP, days after planting; N20, nitrous oxide; m/z, mass to charge ratio; bp, boiling point. compound evolved was NO, but based on results with boiled leaf discs he implied that an enzymic reaction was responsible for the NO(x) evolution. Additional evidence for an enzymic reaction was provided by the isolation of a mutant soybean line (nr...

Nitrate reductase activity of soybeans (Glycine max L. Merr.) was evaluated in soil plots and outdoor hydroponic gravel culture systems throughout the growing season. Nitrate reductase profiles within the plant canopy were also established. Mean activity per gram fresh weight per hour of the entire plant canopy was highest in the seedling stage while total activity (activity per gram fresh weight per hour times the total leaf weight) reached a maximum when plants were in the full bloom to midpod fill stage. Nitrate reductase activity per gram fresh weight per hour was highest in the uppermost leaf just prior to full expansion and declined with leaf positions lower in the canopy. Total nitrate reductase activity per leaf was also highest in the uppermost fully expanded leaf during early growth stages. Maximum total activity shifted to leaf positions lower in the plant canopy with later growth stages.Nitrate reductase activity of soybeans grown in hydroponic systems was significantly higher than activity of adjacent soil grown plants at later growth stages, which suggested that under normal field conditions the potential for nitrate utilization may not be realized. Nitrate reductase activity per gram fresh weight per hour and nitrate content were positively correlated over the growing season with plants grown in either soil or solution culture. Computations based upon the nitrate reductase assay of plants grown in hydroponics indicated that from 1.7 to 1.8 grams N could have been supplied to the plant via the nitrate reductase process. The harvested seed contained 1.1 to 1.2 grams N per plant. Thus, based on previous estimates of approximately 32% of the final N distribution being in the vegetative plant parts, the estimated input of reduced nitrogen via the enzyme assay was in agreement with the actual N accumulation.The amount of calculated N2-fixation by nodules per season with plants grown in hydroponics was less than 2 % of the computed nitrate reduced via leaf nitrate reductase. Thus, the level of nitrate in the nutrient solution appeared to be quite inhibitory to N2-fixation.The response of the soybean plant to nitrogen is confounded by the ability of the plant to utilize both nitrate and N2. Nitrate is considered the primary source of nitrogen available from the soil. The uptake of nitrate and subsequent reduction by nitrate reductase is the primary pathway of soil nitrogen utilization. The utilization of N2 through the symbiotic relationship with Rhizobium japonicum (Kirchner) Buchanan affords a second major pathway of nitrogen input to soybeans.Nitrogen fixation is normally initiated in soybeans 20 to 30 days after planting (3). Thus, initial nitrogen requirements must be met through utilization of nitrogen from the seed and nitrogen from the soil. Recent estimates indicate that some 25 to 30% (80-110 kg N per hectare per season) of the total plant nitrogen was supplied through the N2-fixation process in soybeans as measured with the acetylene-reduction technique (3, 4). It has also been estim...

Studies were conducted to quantitate the evolution of nitrogen oxides (NO(.)) from soybean GlGycine max (L.) Merr.j leaves during in vivo nitrate reductase (NR) assays with aerobic and anaerobic gas purging. Anaerobic gas purging (N2 and argon) consistently resulted in greater NO(.) evolution than did aerobic gas purging (air and 02). The evolution of NO(.) was dependent on gas flow rate and on N02-formation in the assay medium; although a threshold level of N02-appeared to exist beyond which the rate of NO(.) evolution did not increase further. (L.) Merr.] the in vivo NR assay has been optimized for leaves (10,13,17) and roots (7), and the assay has been used to compare estimated reduced N input with actual (Kjeldahl) reduced N accumulation (7, 9, 11). One notable difference between optimized in vivo NR assays of soybean roots and leaves was that roots required N2 purging during assay (7), while N2 purging of leaves during assay resulted in inconsistent results, ranging from slight stimulation to marked inhibition of nitrite accumulation (unpublished data).It has been shown that air purging of herbicide-treated soybean leaves results in evolution of NO and NO2 (collectively NO(.)) (16). The reaction mechanism(s) leading to NO(.) evolution are unknown, although Klepper (16) has proposed that accumulated N02 reacts with plant metabolite(s) to form NO and NO2.Nitrogen oxide (NO) seems to be the primary gas form evolved (16), since NO2 is known to be readily soluble in aqueous solution, forming N02-and NO3&; the equilibrium of the latter two ions being dependent on solution conditions (1).The above observations suggested that similar evolution of NO(x) gasses may be occurring during gas purging of the in vivo l Abbreviations: NR, nitrate reductase; NO(X), refers collectively to nitric oxide (NO) and nitrogen dioxide (NO2); DAP, days after planting. NR assay medium. In addition, it was expected that N2, air, and 02 purging of the in vivo NR assay medium would result in differential accumulation of N02-, due to a more complete blockage of further reduction of N02-to NH4+ under more anaerobic conditions. The objectives of this study were to (a) establish why N2 purging of the in vivo NR assay medium resulted in inconsistent N02 accumulation relative to no gas purging, and (b) determine if evolution of NO(.) gasses under anaerobic and aerobic gas purging of the in vivo NR assay medium could account for the decrease in nitrite accumulation during NR assays of physiologically young soybean leaves. Instruments Co., Lincoln, NE) for a 14-h light period at 27 to 30°C; nighttime temperatures ranged from 17 to 21°C. Plants were sampled at intervals from 11 to 25 DAP for analysis of leaf NR activity. Twenty seedlings were harvested at each sampling time and leaflets from respective nodes were composited and subsampled, 20 leaf discs (1 cm diameter) for each sample (=0.2 g tissue). MATERIALS AND METHODS Plant Growth and SamplingGrowth

It is not known whether NO−3 inhibition of nodulation is primarily due to effects on the host plant or on the bacterials train. This study was conducted to evaluate the effect of NO−3 on nodule appearance and development of nitrogenase (acetylene reduction) activity of various Glycine max (L.) Merr. cultivar ✕ Bradyrhizobiumja ponicum (Kirchner) Buchanan strain combinations. Plants were grown hydroponically in controlled environment cabinets. NO−3 was either resupplied daily (“maintained”) during the growth period or allowed to deplete with plant growth (“rundown”). Nodule development was delayed all NO−3 treatment conditions (both maintained and rundown), the delay being more severe at higher NO−3 concentrations. The external concentration of NO−3 rather than the rate of NO−3 uptake appeared to have a major effect on the initial stages of nodulation. Treatment conditions providing similar rates of NO−3 up take from different solution NO−3 concentrations( 4.0 mM rundown vs. 0.5. mM maintained) resulted in a more marked inhibition of nodule appearance at the higher level of solution NO−3. Maintaining the solution concentration of NO−3 at 1.0 mM following the appearance of nodules greatly retarded, or prevented, the development of nitrogenase activity (C2H2 reduction). Minor differences in the tolerance of nodulation to NO−3 were observed among eight B. j. uponicum strains (preselected from 46 strains). More variation existed among 12 soybean cultivars (preselected from 23 cultivars) with respect to their ability to nodulate in the presence of NO−3. The cvs. Elf and Avoyelles were notable in having good nodulation tolerance to NO−3 when infected by USDA110. This indicated that, with selection and breeding, enhanced NO−3 tolerance of nodulation between soybean and B. japonicum should be possible.

(14), is laborious and not always reliable, as discussed later. While dialysis removes excess NADH (6), it requires specialized equipment. Enzymatic oxidation is also satisfactory (5), except for time and expense. Phenazine methosulfate rapidly oxidizes NADH (2, 7, 8), but it has not been used to oxidize excess NADH in the NR assay.The objectives of this study were to test the effectiveness of PMS in the oxidation of excess NADH in the NR assay medium before color development and to determine if PMS could be used in conjunction with zinc acetate to improve NO2-recovery in the NR assay.The most sensitive method for assaying nitrate reductase in plant extracts is by colorimetric measurement of the product, nitrite (12). Nitrite is diazotized with sulfanilamide and then reacted with N-(l-naphthyl)ethylenediaminedihydrochloride to produce the azo dye which is measured spectrophotometrically at 540 nm. However, excess NADH from the reaction medium interferes with the full development of color (9) MATERIALS AND METHODS Corn (Zea mays L.) and soybeans (Glycine max L. Merr.) were used as the source of NR for these studies. Leaves from corn seedlings, variety "Oh43 X B14", were harvested after 4 to 5 hr of illumination, and NR was extracted and assayed by methods similar to those described by Hageman and Hucklesby (3). The exceptions were that the fresh leaf tissue was ground in a TenBroeck homogenizer in 10 volumes of extraction medium. Standard (reference) assays, containing 50 ,umoles of potassium phosphate (pH 7.5), 20 ,umoles of KNO,, 0.8 ,umole of NADH, and 0.2 ml of the crude extract in a final volume of 4 ml, were incubated 15 min at 30 C. Blank assays were identical except NADH was omitted. The reactions were terminated by boiling for 2 min, and NO,-color was developed by adding an equal volume of a 1